acid phenol extractions
c432752 at showme.missouri.edu
Wed Feb 19 05:08:46 EST 1997
>...tried an acid phenol extraction of a plasmid prep...
>...The yield is abysmal....
Which plasmid were you trying to isolate?
1. One possibility is that the plasmid contains a tightly-bound
"relaxation complex". These enzymes, once "activated" by denaturing agents
(including acid phenol ??), nick plasmid DNA. Their biological role is to
nick the plasmid to enable the conjugal transfer of ssDNA. ColE1-like
plasmids have relaxation complexes; but the respective DNA target, and/or
the genes encoding the relaxation enzyme and its accessory proteins, have
been removed from most of the popular cloning vectors, so that relaxation
complexes are probably not an issue in such cases.
2. Acidic conditions (such as acid phenol ??) accelerate the rate of
spontaneous depurination. Depurinated deoxyribose residues are now
sensitive to a spontaneous internal cleavage reaction. This nicking at
depurinated sites is further accelerated under alkaline conditions.
Therefore, exposing supercoiled plasmids to alkaline conditions after a
pre-exposure to acidic conditions is potentially dangerous. Depurination
followed by nicking becomes more of an issue with larger plasmids, while
most cloning vectors are small.
I have no experience with acidic phenol extractions, so I don't know if
these will work: Since Ed wrote that he equilibrated the phenol at a pH
that may have been <a little below> 4.0, a more precise pH control may be
needed. Do it on ice. Speed it up.
Sincerely, Ben Haskell
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